We propose to exploit the site specific bacterial restriction endonucleases and methylases to study DNA-protein interactions, translation of eucaryotic DNA in a procaryotic organism, and the structure and function of the SV40 tumor virus genome. Site-specific DNA-protein recognition will be investigated using the interaction of EcoRI endonuclease and methylase with DNA and synthetic substrates as the model system. Structural, compositional, and sequence analysis of these two homogenous proteins will be carried out using standard methods of protein chemistry. In vitro recombination of DNA's from diverse sources mediated by ligation of the single stranded cohesive ends generated by restriction endonucleases (EcoRI, HindIII, EcoRII) and subsequent cloning of the molecules will be used to investigate viral and plasmid DNA structure. Translation will be studied in mini-cells after transformation with the appropriate DNA. Similar methods will be used to transform human galactosemic fibroblasts with bacterial gal operon - SV40 hybrid molecules. Specific restriction fragments of SV40 DNA will be used in hybridization experiments to study the patterns of transcription and processing in SV40-transformed cells. Experiments in progress to map ts SV40 mutants will be continued using infection with heteroduplex molecules constructed in vitro from different portions of the genome with ts and wild type DNA restriction fragments. BIBLIOGRAPHIC REFERENCES: Garfin, D. E., Leong, J.A.C., and Goodman, H.M. The EcoRI Site of Simian Virus 40 Deoxyribonucleic acid: Nucleotide Sequences of the Minus and Plus Strands. Biochem. Biophys. Res. Comm. 68, 369-374, 1976. Brown, W. M., Watson, R. W., Vinograd, J., Tait, K. M., Boyer, H. W., and Goodman, H. M. The Structure and Fidelity of Replication of Mouse Mitochondrial DNA-pSCl0l EcoRI Recombinant Plasmids Grown in Escherichia coli K12. Cell 7, 517-530, 1976.